Technical Help for NTA-Ni(II)-Nanogold®

Updated: September 5, 2003 On this page Reducing background binding Eliminating aggregation Other Resources Nanoprobes e-Newsletter Try our e-mail newsletter for tips and tricks for better gold labeling, and to keep up with new applications. Guide to Gold Cluster Labeling Detailed description of gold cluster labeling, tips and tricks for successful conjugations, isolation of conjugates, and how to calculate labeling. MSA Microscopy Listserver and Archives (view or search). Confocal Listserver and Archive (SUNY at Buffalo). Tips and Tricks (from the University of Florida ICBR EM core lab).

Background Binding

When using Ni-NTA-Nanogold®, you should keep in mind that the nature of the binding interaction is different from that of antibodies or other targeted biomolecules such as streptavidin, and therefore different reagents and conditions will be appropriate for reducing undesirable background interactions. Binding occurs by coordination of electronegative atoms, usually aromatic nitrogen, to the nickel (II) ion; if you are localizing or detecting a target that contains other aromatic nitrogen atoms besides the polyhistidine tag (for example, other histidine residues), these may also bind, but the undesirable interaction may be eliminated by treatments that block or disrupt this interaction.

To prevent or eliminate background, we find that the following steps may be useful:

• Wash with a buffer containing imidazole. Imidazole is the active coordinating group in histidine; treatment with imidazole will displace isolated single histidine binding, but it will disturb polyhistidine binding much less because the multiple coordinated histidines increase binding through the chelate effect. Try increasing the imidazole concentration from 10 mM progressively to 200 mM until background is controlled to your satisfaction.

• Increase the ionic strength of the solution. Try 300 mM NaCl, and, if your biomolecule can tolerate it, increase to 1.0 M if necessary. The nitrilotriacetivc acid moiety is negatively charged, and may interact with positively charged regions of a target; increasing the ionic strength will help to prevent this.

• Other possible factors include:
  • hydrophobic interactions, which may be reduced or eliminated by the addition of detergents; try 0.05 % Tween-20, and increase to 0.1 % if necessary.
  • Depending on your specific system, it may also be helpful to vary the pH to find an optimum pH at which charge interactions such as those mentioned above are reduced.
  • Transition metals may also promote interactions bwteen the NTA group and electronn donating groups in your specimen. Washign with 0.05 M disodium ethylene diamine tetraacetic acid (EDTA) will help to remove these.
  • Interaction of thiols (sulfhydryls) with gold. Thiols have a strong affinity for gold, and if they are present in your specimen, any gold particle species, including Ni-NTA-Nanogold®, may bind to them. Avoid the use of reducing buffers or preservatives containing thiols, such as dithiothreitol (DTT), mercaptoethanol, or mercaptoethylamine hydrochloride. If your specimen contains exposed thiols, they may be blocked with N-ethylmaleimide.

If you are seeing background signal after silver or gold enhancement, a number of methods are available for stopping the reaction, or preventing further reaction after the desired end-point by reagents that have diffused into specimens.

Silver and Gold Enhancement "Stop" Reactions and back development:

Under most circumstances, repeated washing with deionized or distilled water will be sufficient to halt the silver enhancement process. However, it may continue within the specimen, and this can sometimes give over-development or a dark appearance in the light microscope. In this case, one of the following methods may be used to "stop" the silver enhancement reaction:

• 1% acetic acid.
• 1% acetic acid followed by photographic fixer (Agefix, Agfa-Gevaert, or Ilfospeed 200, Ilford).
• direct photo fix, using those just mentioned.
• brief rinse in 2.5% sodium chloride.
• 15-25% aqueous sodium thiosulfate plus 15% sodium sulfite.
• 1% acetic acid, washes in acetate buffer, toning in 0.05% HAuCl₄ 3-10 min, with excess silver removed with 3% sodium thiosulfate. We found that Nanogold-labeled proteins run on a polyacrylamide gel kept low backgrounds when stopped with 10% acetic acid with 10% glucose in water, as opposed to just a water stop.

These methods, and their references, are discussed in our recent review of Nanogold® technology:

• Hainfeld, J. F., and Powell, R. D.: Nanogold Technology: New Frontiers in Gold Labeling. Cell Vision, 4, 408-432 (1997).

  Complete paper (PDF) (courtesy of the Cell Vision).

In the event that the reaction has proceeded too far, it may be "back-developed" to remove the excess background staining by treatment with Farmer's solution (0.3 ml 7.5% potassium ferricyanide, 1.2 ml of 20% sodium thiosulfate, 60 ml water). Application of this solution briefly to your sample before gold toning may help to remove backgroud silver deposition.

Reference:

Danscher, G.: Histochemical demonstration of heavy metals. A revised version of the silver sulphide method suitable for both light and electron microscopy. Histochemistry, 71, 1-16 (1981).

• Abstract (courtesy of PubMed (Medline)).

Eliminating Aggregation

In the development of Ni-NTA-Nanogold®, it was found that a form containing multiple NTA-Ni(II) groups produced the best overall combination of labeling selectivity, density and sensitivity. However, because this can interact with polyhistidine tags on several protein molecules simultaneously, it is possible that it will act to aggregate proteins in solution, or perturb the formation of protein complexes in suspension. The best aproach to preventing this is to select a ratio of reagent to protein such that the stoichiometry reduces or eliminates this possibility. For example, if your protein has only one polyhistidine tag, then using an excess of the Ni-NTA-Nanogold® reagent will guard against the possibility of multiple interactions. You can also help avoid the possibility by carefully selecting when to add the reagent, for example after complex assembly.

The methods given above for reducing non-specific background binding may also help to reduce aggregation. Using reduced concentrations of target proteins may also help.

Contents | Catalog Info: Ni-NTA-Nanogold® | Catalog (Distributor) | References for Ni-NTA-Nanogold® | News

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